Methods for treating malignancies using coxsackieviruses

ABSTRACT

There is a disclosed a method of killing abnormal cells such as malignant cells including melanoma cells, using a virus recognising at least one of a cell adhesion molecule and a complement regulatory protein. The virus may be a member of the Picornaviridae family. Coxsackie A-group viruses have been found to be particularly suitable. The cell adhesion molecule is desirably a member of the immunoglobulin (Ig) superfamily. Typically, the complement regulatory protein will be DAF.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 10/148,008 filed Nov. 5, 2002, now issued as U.S. Pat. No.7,361,354; which is a 35 USC §371 National Stage application of PCTApplication No. PCT/AU00/01461 filed Nov. 27, 2000; which claims thebenefit under 35 USC §119(a) to Australia Patent Application No. PQ 4256filed Nov. 25, 1999. The disclosure of each of the prior applications isconsidered part of and is incorporated by reference in the disclosure ofthis application.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the killing of abnormal cells utilisinga virus. There is also described a method of screening cells toascertain whether they are susceptible to treatment with the virus, aswell as pharmaceutical compositions incorporating the virus. Theinvention finds veterinary use as well as broad application in the humanmedical field.

2. Background of the Invention

Melanoma is a leading cause of morbidity in the human population.Australia has the highest rate of melanoma in the world. Melanoma is anaggressive skin cancer and is the third most common cancer in Australiafor both men and women. It is predicted that one in thirty Australianshave a form of melanoma resulting in the death of more than one thousandpeople per year in that country alone. When detected early most forms ofmelanoma can be effectively treated. However, the control of moreadvanced forms is less successful and an area of intensive research. Amajor goal in this area of research is the identification of moleculesthat are differentially expressed in benign and malignant melanocytictumours that can be used for diagnosis and as targets for anti-cancertherapies (Kageshita T. et al; 1993).

Intercellular adhesion molecule-1 (ICAM-1), a crucial molecule incellular inflammatory interactions, is an accepted melanoma progressionantigen. Surface-expression of ICAM-1 on melanomas has been highlycorrelated with malignant melanoma progression (Kraus A. et al; 1997 andMorandini R. et al; 1998).

ICAM-1 is a member of the immunoglobulin (Ig) superfamily and a counterreceptor for the integrin leucocyte function antigen-1 (LFA-1/CD11a) andMac-(CD11b), and is a cellular attachment molecule for 90% of humanrhinoviruses (Stuanton D. E., et al; 1989). In addition, ICAM-1 plays animportant role in the pathogenesis of not only rhinovirus infection, butalso in Plasmodium falciparum infection and in the exacerbations ofasthma, chronic bronchitis and cystic fibrosis. Recently, complementregulatory proteins have been reported to be up-regulated on the surfaceof malignant melanomas, in particular decay-accelerating factor known asDAF (Cheung N K et al; 1998).

Viruses capable of inducing lysis of malignant cells through theirreplication process are known as oncolytic viruses and trials usingoncolytic viruses to treat malignancies have been performed (NemunaitisJ; 1999). Most oncolytic viruses require proliferation in the samespecies or cell lineage. Infection of a cell by a virus involvesattachment and uptake into the cell which leads to or is coincidentalwith uncoating of the viral capsid, and subsequently replication withinthe cell (Fenner F., et al. The Biology of Animal Viruses. AcademicPress. New York, 1974 Second Ed.)

Oncolytic viruses assessed for capacity to kill cancer cells haveincluded the adenovirus subtype Egypt 101 virus which showed oncolyticactivity in the HeLa uterine/cervix cancer cell line, mumps virus fortreatment of gastric carcinoma, uterine carcinoma and cutaneouscarcinoma, Newcastle Disease Virus (NDV), influenza virus for treatmentof ovarian cancer, and adenovirus for treatment of for instance,cervical carcinoma (Nemunaitis J; 1999). Other reports have indicatedthat adenoviruses and attenuated poliovirus recombinants may have use inthe treatment of malignant glioma cells (Alemany R., et al 1999;Andreansky S. S., 1996), and that reovirus shows lytic capability inhuman U87 glioblastoma cells and NIH-3T3 cells with an activated Rassignalling pathway (Coffey M. C, et al, 1998; Strong J. E. et al, 1998).

In addition, a vaccinia oncolysate has been used in clinical trials totreat melanoma (Stage II) patients (Nemunaitis J., 1999), Modified,non-neurovirulent Herpes simplex viruses (HSV) have also been reportedas showing promise for the treatment of brain tumours includingintracranial melanoma, and subcutaneous human melanoma (Randazzo B. R.,1997), while adenovirus infection has been reported to enhance killingof melanoma cells by the plant mitotoxin, saporin (Satyamoorthy K.,1997).

The receptor on target cells recognised by adenovirus differs fordifferent adenovirus types. That is, adenovirus subgroups A, C, D, E andF for instance recognise the CAR receptor while Adenovirus type 5(subgroup C), Adenovirus type 2 (subgroup C) and Adenovirus type 9(subgroup D) recognise major histocompatibility class II molecule,α_(m)β₂ and α_(v) integrins, respectively. The CAR receptor is known tobe expressed on melanoma cell lines (Hemmi S., et al, 1998). Heparansulfate is recognised by Herpes simplex types 1 and 2 and human herpesvirus 7, Adeno-associated virus type 2. The receptor for humanHerpesvirus 7 is CD4 while Epstein-Barr virus recognises complementreceptor Cr2 (CD21). Poliovirus type 1 and 2 recognise poliovirusreceptor (Pvr) for cell adhesion while reovirus recognises sialic acid.Influenza A and B virus recognise the sialic acid N-acetyl neuraminicacid for cell adhesion. In contrast, influenza type C virus recognisesthe sialic acid 9-O-acetyl neuraminic acid. Vaccina virus recognisesboth epidermal growth factor receptor and heparan sulfate.Coxsackievirus A13, A15, A18 and A21 recognise ICAM-1 and the complementregulatory protein DAF (CD55) (see eg. Shafren D. R., et al 1997). DAFis also recognised by Enterovirus 70. See for instance Flint S J, et al(2000) Principles of Virology: molecular biology, pathogenesis andcontrol. ASM Press, Washington.

Metastatic tumour spread is a pathological process associated with aseries of adhesion/de-adhesion events coupled with regulated tissuedegradation. It is known that adhesion to and migration through theextracellular matrix is essential for tumour invasion. The largestfamily of extracellular adhesion molecules is the integrin family(Marshall J. F. and Hart I. R., 1996) and members of the αvβ group ofintegrins have been shown to be expressed on a variety of cell types.For instance α_(v)β₁ is expressed on neuroblastoma, melanoma andosteosarcoma cells, α_(v)β₃ is expressed on melanoma, glioblastoma andrenal carcinoma cells, and α_(v)β₅ is expressed on melanoma cells as isα_(v)β₈ (Marshall J. F. and Hart I. R., 1996).

Despite progress being made in the treatment of malignancies, thetreatment of cancer including melanoma presents a major challenge forresearch and there remains the need for alternatives to existing therapyapproaches.

SUMMARY OF THE INVENTION

The present invention stems from the surprising finding of significantkilling of abnormal cells can be achieved with the use of a virus andthe recognition/interaction of cell expressed markers utilised by thevirus for infectivity of the cells.

In one aspect there is provided a method of treating abnormal cells in amammal comprising administering to the mammal an effective amount of avirus capable of infecting the abnormal cells whereby death of the cellsis caused and which recognises at least one of a cell adhesion moleculeof the immunoglobulin (Ig) superfamily and a complement regulatoryprotein for infectivity of the abnormal cells.

The term “abnormal cells” for the purpose of the present invention is tobe taken in a broadest sense to include malignant cells, the cells ofany abnormal growth and any cells having abnormal upregulated expressionof at least one of the cell adhesion molecule and the complementregulatory protein relative to corresponding normal cells of the samecell type expressing their normal phenotype, whether the cells arecancer cells or not and whether the cells proliferate at an abnormalrate or not. Accordingly, the term encompasses pre-neoplastic andneoplastic cells, and non-cancer cells that may or may not ultimatelydevelop into cancer cells. An abnormal growth may for instance be abenign or malignant tumour. Typically, the abnormal cells will bemalignant cells and usually melanoma cells.

Generally, the expression of at least one of the cell adhesion moleculeand the complement regulatory protein will be upregulated compared tosurrounding tissue in which the abnormal cells are found.

Hence, the virus will typically preferentially infect the abnormal cellsdue to the greater likelihood of contacting at least one of the celladhesion molecule and complement regulatory protein on those cells. Assuch the virus may be used to effectively target the abnormal cells.

In another aspect of the invention there is provided a method oftreating melanoma in a mammal comprising administering to the mammal aneffective amount of a virus capable of infecting melanoma cells wherebydeath of the cells is caused and wherein the virus recognises at leastone of a cell adhesion molecule and a complement regulatory protein forinfectivity of the melanoma cells.

The virus may also be used to screen cells to ascertain for instancewhether the virus may be suitable for treating the patient from whichthe cells were obtained or whether a different treatment protocol notinvolving the virus may be more beneficial to the mammal. Conversely,different viruses may be screened using samples of cells taken from thepatient in order to select the most appropriate virus for treating themammal.

Accordingly, in another aspect of the invention there is provided amethod of screening abnormal cells for determining whether the cells aresusceptible to viral induced cell death, comprising the steps of:

(a) providing the abnormal cells;

(b) adding to the cells an effective amount of a virus which recognisesat least one of a cell adhesion molecule of the immunoglobulin (Ig)superfamily and a complement regulatory protein for infectivity of theabnormal cells;

(c) incubating the abnormal cells in the presence of the virus for aperiod of time; and

(d) determining whether the virus has infected and caused death of atleast some of the abnormal cells.

In a further aspect of the present invention there is provided a methodof screening melanoma cells for determining whether the cells aresusceptible to viral induced cell death, comprising the steps of:

(a) providing the melanoma cells;

(b) adding to the melanoma cells an effective amount of a virus whichrecognises at least one of a cell adhesion molecule and a complementregulatory protein for infectivity of the melanoma cells;

(c) incubating the melanoma cells in the presence of the virus for aperiod of time; and

(d) determining whether the virus has infected and caused death of atleast some of the melanoma cells.

A virus may be selected for use in a method of the invention by testingwhether a given virus is capable of infecting and causing the death ofabnormal cells expressing at least one of the cell adhesion molecule andthe complement regulatory protein. In particular, the testing mayinvolve screening a number of different viruses by incubating each viruswith a sample of the abnormal cells respectively, and determiningwhether the cells are killed as a result of infection.

Accordingly, in another aspect of the present invention there isprovided a method of testing whether a virus is capable of infectingabnormal cells whereby death of the cells is caused and which recognisesat least one of a cell adhesion molecule of the immunoglobulin (Ig)superfamily and a complement regulatory protein for infectivity of theabnormal cells.

In a further aspect of the present invention, there is provided a methodof testing whether a virus is capable of infecting melanoma cellswhereby death of the cells is caused and which recognises at least oneof a cell adhesion molecule and a complement regulatory protein forinfectivity of the melanoma cells.

In still another aspect of the invention there is provided a method ofscreening a virus for ability to infect and cause death of abnormalcells, comprising the steps of:

(a) selecting a virus which recognises at least one of a cell adhesionmolecule of the immunoglobulin (Ig) superfamily and a complementregulatory protein for infectivity of the abnormal cells;

(b) incubating the selected said virus with a sample of the abnormalcells for a period of time; and

(c) determining whether the selected said virus causes death of at leastsome of the abnormal cells.

In another aspect of the present invention there is provided a method ofscreening a virus for ability to infect and cause death of melanomacells, comprising the steps of:

(a) selecting a virus which recognises at least one of a cell adhesionmolecule and a complement regulatory protein for infectivity of themelanoma cells;

(b) incubating the selected said virus with a sample of the melanomacells for a period of time; and

(c) determining whether the selected said virus causes death of at leastsome of the melanoma cells.

The method may also comprise the step of comparing the ability of theselected virus to infect and cause the death of the cells with that ofanother virus subjected to steps (b) and (c) utilising another sample ofthe cells.

Death of the cells following infection with the virus may result fromeither lysis of the cells due to intracellular replication of the virusor due to the infection triggering apoptosis most likely as a result ofthe activation of cellular caspases.

Once lysed, the cytosolic contents of infected cells spills from theruptured plasma membranes, and antigens capable of eliciting an immuneresponse to the abnormal cells may be released. Hence, treatment ofabnormal cells in a mammal in accordance with a method of the inventionmay provide a boost to the immunity of the mammal against the abnormalcells.

Accordingly, in another aspect of the invention there is provided amethod of inducing an immune response in a mammal comprising infectingabnormal cells in the mammal with a virus whereby death and lysis of thecells is caused with release of antigens therefrom for generation ofsaid immune response, wherein the virus recognises at least one of acell adhesion molecule of the immunoglobulin (Ig) superfamily and acomplement regulatory protein for infectivity of the abnormal cells.

In yet another aspect of the present invention there is provided amethod of inducing an immune response in a mammal against melanomacells, comprising infecting the melanoma cells in the mammal with avirus whereby death and lysis of the cells is caused with release ofantigens therefrom for generation of said immune response, wherein thevirus recognises at least one of a cell adhesion molecule and acomplement regulatory protein for infectivity of the melanoma cells.

Generally, the virus will be provided in the form of a pharmaceuticalcomposition for use in a method of the invention. As such, in a yetfurther aspect of the invention there is provided a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier togetherwith a virus capable of infecting abnormal cells whereby death of thecells is caused and which recognises at least one of a cell adhesionmolecule of the immunoglobulin (Ig) superfamily and a complementregulatory protein for infectivity of the abnormal cells.

In still another aspect of the present invention there is provided apharmaceutical composition comprising a pharmaceutically acceptablecarrier together with a virus capable of infecting melanoma cellswhereby death of the cells is caused and which recognises at least oneof a cell adhesion molecule and a complement regulatory protein forinfectivity of the melanoma cells.

In another aspect is provided the use of the pharmaceutical compositionin a method of the invention.

In a further aspect of the invention there is provided the use of avirus in the manufacture of a medicament for treating malignant cells,wherein the virus is capable of infecting the abnormal cells wherebydeath of the cells is caused and which recognises a cell adhesionmolecule of the immunoglobulin (Ig) superfamily for infectivity of saidabnormal cells.

In another aspect of the present invention there is provided the use ofa virus in the manufacture of a medicament for treating melanoma whereinthe virus is capable of infecting melanoma cells whereby death of thecells is caused and which recognises at least one of a cell adhesionmolecule and a complement regulatory protein for infectivity of themelanoma cells.

In addition, there is provided delivery means for being held against theskin of a mammal for facilitating delivery of the virus to the mamma!,and which is impregnated with a pharmaceutical composition of theinvention for contact with the skin, when the delivery means is heldagainst said skin of the mammal in use. Generally, the delivery meanswill be adapted to enable it to hold in position over the skin at thedesired site of treatment.

Preferably, the virus will be capable of binding to or otherwiseassociating with both the cell adhesion molecule and the complementregulatory protein. The complement regulatory protein will usually forma complex with the cell adhesion molecule or have a close spatialassociation with the cell adhesion molecule, and enhance the ability ofthe virus to infect the abnormal cells. Preferably, the complementregulatory protein will be decay-accelerating factor (DAF).

Preferably, the cell adhesion molecule is a member of the immunoglobulin(Ig) superfamily which includes V-CAM-1 and the intercellular adhesionmolecules ICAM-1, ICAM-2 and ICAM-3. Preferably, the cell adhesionmolecule is ICAM-1.

Normally, the virus will be an animal RNA virus and typically, anon-enveloped RNA virus with an icosohedral capsid and a single RNAstrand genome.

Preferably, the virus will be a member of the Picornaviridae family.Members of the immunoglobulin (Ig) superfamily have a plurality ofextracellular domains and the virus will desirably interact with theoutermost domain closest to the N-terminus of the immunoglobulin (Ig)superfamily molecule. Preferably, the virus will be from the genusEnterovirus and most preferably, the virus will be a Coxsackievirus.Coxsackievirus is a human enterovirus and most enteroviral infections,even with the more virulent members of the group, cause few or noclinical symptoms. CAV21 infection for instance is associated withdevelopment of common colds and infantile diarrhoea.

Hence, in another aspect of the present invention there is provided amethod of treating abnormal cells in a mammal comprising administeringto the mammal an effective amount of a Coxsackievirus.

In a still further aspect of the present invention there is provided amethod of treating melanoma in a mammal comprising administering to themammal an effective amount of a Coxsackievirus.

Typically, the Coxsackievirus will be a Coxsackie A-group virus, andwill normally be selected from the group consisting of Coxsackievirusesserotypes 1 to 24 (CAV1-24), and most preferably from CAV13, CAV15,CAV18 and CAV21.

While the virus will usually be a common animal virus the invention isnot limited thereto and a recombinant virus engineered to be capable ofinfecting and causing the death of the abnormal cells, or a virus thathas otherwise been modified to enhance its ability to infect the cellsand cause the death of the cells post infection, may be utilised. Forinstance, the virus may be modified to recognise additional celladhesion molecules such as α_(v)β₃, α_(v)β₅ or α_(v)β₆.

Moreover, the same virus may be administered to the mammal duringdifferent treatment courses. Preferably, however, different viruses areused for different treatment courses to avoid or lessen the potentialeffect of any immune response to the previous virus administered. Thevirus may for instance be administered topically, intratumourally orsystemically to the patient.

The mammal may be any mammal suffering from a malignancy and in need oftreatment. Preferably, the mammal will be a human being.

A method of the invention may be used as an adjunct to conventionalcancer treatment or as a treatment in the absence of other therapeutictreatments. In particular, a method of the invention may be utilisedwhere conventional treatment is not suitable or practical, or in theinstance where excision of abnormal cells may leave scaring ordisfigurement which is unacceptable to the patient, particularly thepatient's face such as from their nose or lip. Alternatively, the virusmay be administered to the patient prior to and/or immediately afterexcision of abnormal cells.

Accordingly, the instant methods provide an alternative therapeutictreatment which may be used both following diagnosis of early stage andlatter stage malignancy, and which further finds application for killingcells prior to and remaining after surgery.

Using protocols as described herein the skilled addressee will be ableto readily select a suitable virus for use in the methods of theinvention, and determine which abnormal cells are susceptible toinfection leading to the death of the cells. The abnormal cells may forinstance be prostate cancer cells, breast cancer cells, stomach cancercells, gastric carcinoma cells, colon cancer cells, colorectal cancercells, glioma cancer cells, skin cancer cells or other malignant cells.

A method of the invention is particularly suitable for treating amalignancy of the skin or a malignancy that has spread from the skinsuch as melanoma.

Unless the context clearly requires otherwise, throughout thedescription and the claims, the words ‘comprise’, ‘comprising’, and thelike are to be construed in an inclusive sense as opposed to anexclusive or exhaustive sense; that is to say, in the sense of“including, but not limited to”.

The invention will now hereinafter be further described with referenceto a number of non-limiting preferred embodiments.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1 shows immunoperoxidase staining of surface ICAM-1 expression onmelanoma cells. ICAM-1 expression (white arrows) is indicated by darkcell staining;

FIG. 2 shows relative levels of ICAM-1 and DAF expression by themelanoma cell lines Sk-Mel-28 and ME4405;

FIG. 3 indicates lytic infection of two human melanoma cells lines byCoxsackievirus A21 at different time intervals post infection;

FIG. 4 indicates lytic infection of human melanoma cells from a primarymelanoma induced in a nude mouse with various doses of CoxsackievirusA21;

FIG. 5 indicates lytic infection of preparations of suspension andadherent primary malignant cells from a chest wall melanoma byCoxsackievirus A21 at 20 hours post infection;

FIG. 6 (A) indicates lytic infection of six human melanoma cell lines byCoxsackievirus A21 at twenty-three hours post infection; (B) indicatesresults of flow cytometric analysis of DAF (dark line) and ICAM-1(lighter line) on the surface of human melanoma cells;

FIG. 7 indicates lytic infection of different tumour cell lines byrepresentative human enteroviruses;

FIG. 8 indicates lytic infection of a human melanoma biopsy from lymphnode by human enteroviruses Coxsackievirus A21 and B3;

FIG. 9 indicates lytic infection of prostate cancer cells by selectedCoxsackievirus;

FIG. 10 shows the capacity of CAV21 and CAV15 to specifically lyricallydestroy melanoma cells without infecting non-melanoma cells;

FIG. 11 indicates subcutaneous administration of CAV21 infected cells toNOD-SCID mice inhibits human melanoma tumour formation;

FIG. 12 is a graph showing results of intratumoural treatment ofpreformed Sk-Mel-28 melanoma with CAV21;

FIG. 13 is a graph showing results of intratumoural treatment ofpreformed Sk-Mel-28 melanoma with CAV 15;

FIG. 14 shows Sk-Mel-28 tumours 35 days post inoculation with PBS (lefttumour) and CAV15 (right tumour); and

FIG. 15 is a graph showing the effect of intratumoural treatment ofpreformed ME4405 melanoma with CAV21.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

The following biological material was deposited under the BudapestTreaty on the dates provided below, and is available from the AmericanType Culture Collection (ATCC), 10801 University Blvd., Manassas,Virginia 20110-2209 USA:

Definition: Coxsackie group A virus, strain CVA13

ATCC No.: PTA-8854

Date of Deposit: Dec. 20, 2007;

Definition: Coxsackie group A virus, strain CVA15 (G9)

ATCC No.: PTA-8616

Date of Deposit: Aug. 15, 2007;

Definition: Coxsackie group A virus, strain CVA18

ATCC No.: PTA-8853

Date of Deposit: Dec. 20, 2007;

and

Definition: Coxsackie group A virus, strain CVA21 (Kuykendall)

ATCC No.: PTA-8852

Date of Deposit: Dec. 20, 2007.

To determine whether a virus is capable of infecting and causing deathof cells of a tumour, a biopsy may be taken from the tumour and apreparation of cells prepared using conventional techniques prior to:(i) confirming virus receptor cell surface expression and (ii)challenging the cells with the virus and monitoring the cells forinfection and cell death over a predetermined incubation period,typically about 2 days although this may vary depending on the virusused. A number of viruses may be screened in this way simultaneouslyutilising different aliquot's of the prepared malignant cells, the virusshowing the greater degree of infectivity and cell death may then beselected for administration to the subject from whom the biopsy wastaken. Similarly, different malignant cell preparations from biopsiestaken from different sources may be employed in an assay using aspecific virus. The biopsies may be taken from different sites of asingle individual or from a number of individuals.

A virus used in a method as described herein will desirably cause few oronly minor clinical symptoms in the recipient. Such viruses are readilyobtainable from commercial sources well known to the skilled addresseeand can be screened for their effectiveness in the instant methods inthe manner described above. Desirably, the virus will normally beselected from Coxsackie A-group viruses. CAV21 is preferred and inparticular CAV21 (Kuykendall) (Sickles G. M., Proc. Soc. Exp. Biol. Med.102:742; Shafren D. et al J. Virol 1997, 71:4736; Hughes et al, J. GenVirol. 1989, 70:2943; Schmidt, N. J., et al, Proc. Soc. Exp. Biol. Med.,1961, 107:63. CAV21 (Kuykendall) is available from the American TypeCulture Collection (ATCC) 10801 University Boulevard, Manassas, Va.20110-2209, United States of America under Accession No. VR-850.

For the purpose of simply screening a given virus to ascertain whetherit is capable of infecting and causing the death of malignant cells,malignant cell lines may be used for this purpose rather than primarymalignant cells isolated from a biopsy.

Virus that recognises at least one of ICAM-1 and the complementregulatory protein DAF will typically be used. Besides being expressedon melanoma cells (Cheung N. K. et al 1998), DAF has also been shown tohave upregulated expression on colonic adenocarcinoma cells in situ andon the human colonic adenocarcinoma cell line HT29. The expression ofDAF has been postulated to promote resistance of the cells to complementmediated damage and so represents a possible mechanism of tumour escape(Bjørge L., et al; 1996).

Upregulated expression of ICAM-1 has been reported in a variety ofmalignant cell types including gastric carcinoma and adenoma cells (NasuR., 1996; and Koyama S., 1992), prostrate cancer cells (Rokhlin O. W.,and Cohen M. B., 1995), and human breast cancer cells (Sgagius M. K.,1996). Studies have also shown that V-CAM1 is expressed with ICAM-1 onbreast cancer cells (Regidor P. A., et al; 1998). In addition, IC AM-1is known to be expressed on medullary carcinoma cells (Bacuss S. S. etal; 1994), myeloma cells (Maloney D. G. et al; 1999) and thyroidcarcinoma cells. ICAM-1 positive staining has also been reported inprimary tumours such as papillary adenocarcinoma, and metastatic tumoursfrom brain, liver and the adrenal gland (Fernandez-Real J. M; 1996).

Tumours occurring on the skin such as melanoma are particularly suitablecandidates for treatment with the virus. In instances where melanoma hasspread to lymph nodes, the lungs or other organs, the virus may beadministered to those sites and/or the surrounding tissue as describedabove during a surgical procedure to expose such sites for treatment.

The selected virus will preferably be injected directly into a number ofsites on a malignant tumour in order to maximise the area for potentialinfection of the tumour by the virus. Normally, tissue surrounding thetumour will be injected or otherwise treated with the virus given thepossibility of malignant cells being present in the tissue. If thetumour is not detected until it is relativity advanced, surroundingtissue may be injected with the virus following surgical excision of thetumour itself.

Rather than being injected directly into a malignant tumour, the virusmay be administered systemically by intravenous injection into the bloodstream of the recipient at a location adjacent to the tumour site fordelivery to the tumour. Similarly, the virus may be administeredsubcutaneously, intraperitoneally or for instance, intramuscularly ifdeemed appropriate. Generally, however, direct injection into the tumouris preferred given the possibility of the existence of antibodiesspecific for the virus and thereby the potential decreased efficacy ofalternate such modes of virus delivery.

The virus may also be applied topically to tumours either alone or incombination with direct injection of the virus into the tumour. In thisinstance, the virus may be applied by way of delivery means for beingpressed against the malignant site on the skin to be treated and whichis impregnated with a suitable pharmaceutically acceptable carrier formaintaining the integrity of the virus to allow for infection of themalignant cells by the virus. The delivery means may be in the form offor instance, a patch, a pad, a wad, bandaging or the like suitable forlocalising the virus in the area to be treated. Typically, the deliverymeans will be a patch provided with an adhesive around an undersideperimeter thereof for sticking the patch on the skin and thereby holdingthe patch in the desired position and the inoculant in contact with thepatients skin.

Generally, one or more small incisions will be made into the malignancyand/or surrounding tissue to provide a site of entry for the virus intosame.

The carrier medium used for inoculating the recipient with the virus maybe a fluid such as physiological saline, or any other conventionallyknown medium deemed appropriate such as commercially available gelssuitable for pharmaceutical use and for administering the virus to thesite of treatment.

The inoculant will generally contain from about 1×10² to about 1×10¹⁰plaque forming units per ml of the inoculant. Preferably, the inoculantwill contain greater than about 1×10⁵ plaque forming units per ml ofinoculant. The amount of inoculant administered to the patient may bereadily determined by the attending physician or surgeon in accordancewith accepted medical practice taking into account the general conditionof the patient, the stage and location of the malignancy together withthe overall size and distribution of the area to be treated with thevirus. Typically, the patient will be treated with an initial dose ofthe virus and subsequently monitored for a suitable period of timebefore a decision is made to administer further virus to the patientpending factors such as the response of the patient to the initialadministration of the virus and the degree of viral infection andmalignant cell death resulting from the initial treatment.

Desirably, an individual will be treated with the virus over a period oftime at predetermined intervals. The intervals may be daily or rangefrom 24 hours up to 72 hours or more as determined appropriate in eachcircumstance. The same or a different virus may be administered eachtime to avoid or minimise the effect of any immune response to apreviously administered virus, and a course of treatment may extend forone to two weeks or more as may be determined by the attendingphysician. Most preferably, virus to which the mammal has not previouslybeen exposed or to which the mammal generates a relatively minor immuneresponse as may be determined by standard techniques will beadministered.

While readily available known viruses may be suitably employed in amethod of the invention, a virus modified or engineered usingconventional techniques may also be utilised. For instance, a virus maybe modified to employ additional cell adhesion molecules as cellreceptors. For example, Coxsackievirus A21 may be modified usingsite-directed mutagenesis so that the peptide motif “RGD” is expressedon the viral caspid surface as is the case with Coxsackievirus A9(CAV-9). The RGD motif is recognised by all the α_(v) integrinheterodimers and this capsid modification may for instance allow thevirus to bind the integrin α_(v)β₃, a cell adhesion molecule which hasbeen shown to be upregulated in combination with ICAM-1 on the surfaceof malignant melanoma lesions (Natali P. G.; 1997) leading to enhanceduptake of the virus via interaction with the integrin molecule orsubsequent interaction with ICAM-1. Alternatively, the virus may bemodified to recognise a selectin such as E-selectin.

The invention will now be described with reference to a number ofexamples described below.

EXAMPLE 1

1.1. Cell Lines

Continuous cultures of Rhabdomyosarcoma expressing ICAM-1 cells(RD-ICAM-1), HeLa-B cells, and human lung fibroblast cells (MRC5) weremaintained in Dulbecco's Modified Eagle's Medium (DMEM) and 10% fetalcalf serum (FCS). Two melanoma cell lines Sk-Mel-28 and ME4405 wereobtained from Dr. Ralph (Department of Biochemistry and MolecularBiology, Monash University, Victoria, Australia) and Dr. Peter Hersey,Cancer Research Department, David Maddison Building Level 4, RoyalNewcastle Hospital, Newcastle, New South Wales, Australia, respectively.The cell line Sk-Mel-28 is a metastatic melanoma cell line found to beresistant to chemotherapeutic drugs (56). The melanoma cell cultureME4405 was established from specimens of primary melanoma lesions (69).The two melanoma cell lines were maintained in DMEM containing 10% FCS.Rhabdomyosarcoma cells (RD) a heteroploid human embryonal cell line, andHeLa-B cells an aneuploid cell clone derived from human squamousepithelial cells, were obtained from the Entero-respiratory Laboratory,Fairfield Hospital, Melbourne, Victoria, Australia. RD cells stablytransfected with cDNA encoding the immunoglobulin superfamily moleculeICAM-1 providing the RD-ICAM-1 cell line have been described elsewhere(Shafren D R, et al; 1997). MRC5 cells, derived from human lungfibroblasts were obtained from Bio-Whittaker, USA.

1.2. Viruses

Strains of CAV21 (Kuykendall strain), CAV15 (G-9) and CVB3 (Nancy) wereobtained from Margery Kennett, Entero-respiratory Laboratory, FairfieldHospital, Melbourne, Victoria, Australia.

1.3. Virus Propagation

RD-ICAM-1 cultures (80-95% confluent) were infected with 10⁴ TCID₅₀ (50%tissue culture infectious dose) of Coxsackievirus A strains according tostandard procedures. Infected cells were incubated at 37° C. untilcomplete cytopathic effect was observed (within 2 days). Cells were thenfrozen at −80° C. and thawed to release the remaining intracellularvirus particles. The virus-containing medium was clarified of cellulardebris by centrifugation for 5 min at 1000×g and stored as 500 μlaliquots at −80° C. CVB3 was propagated in HeLa-B cells in the samemanner as described above.

1.4 Monoclonal Antibodies (MAbs)

MAb 1H4 which recognises the third SCR of DAF (24) was a gift from Dr.B. Loveland, Austin Research Institute, Melbourne, Victoria, Australia.MAb WEHI-CAM recognises the first domain of ICAM-1 (Berendt A R, et al;1992) and was provided by Dr. A. Boyd, Walter and Eliza Hall Institute,Melbourne, Victoria, Australia.

1.5. Flow Cytometric Analysis

Cells (1×10⁶) in 100 μl aliquots were incubated with Mab IH4 or MabWEHI-CAM diluted in DMEM containing 1% FCS on ice for 30 min. The cellswere then washed with 5.0 ml of PBS, pelleted at 1,000×g for 5 min andresuspended in 100 μl of fluorescein isothiocyanate-conjugated goatanti-mouse immunoglobulin G (Silenus, Melbourne, Australia) diluted inPBS. Following incubation on ice for 30 min the cells were washed andpelleted, and resuspended in PBS for analysis with a FACStar analyser(Becton Dickinson, Sydney, Australia).

1.6. Colourimetric Infectivity Assay

The stock virus solutions of CAV21 and CAV15 were serially diluted10-fold in DMEM containing 1% foetal calf serum (FCS). RD-ICAM-1 cellmonolayers in 96-well plates were inoculated with 100 μl of serialdilutions of the viruses for 48 h at 37° C. To quantitate cell survival,monolayers were incubated with 100 μl of a crystal violet-methanolsolution (5% w/v crystal violet, 10% v/v methanol, 10% v/v formaldehydesolution in PBS) and washed with distilled water. The plates were readon a multiscan enzyme-linked immunosorbent assay plate reader at awavelength of 540 nm. Fifty percent endpoint titres were calculated(Reed L J and Muench H A; 1938) and expressed as 50% tissue cultureinfectious dose (TCID₅₀) per millilitre. A well was scored positive ifabsorbance was less than three standard deviations of the no-viruscontrol. The TCID₅₀ for CAV21 was determined to be 2.7×10⁴ units per mlwhile for CAV15, the TCID₅₀ was determined to be 1.6×10⁴ units per ml.

1.7. Surface Expression of ICAM-1 and DAF

The relative levels of ICAM-1 and DAF expression on the surface of themelanoma cell lines SK-Mel-28 and ME4405 was determined by flowcytometric analysis. The results are shown in FIG. 2.

As can be seen, flow cytometric analysis revealed comparable high levelICAM-1 and DAF expression on the surface of the two melanoma cell lines.A further 6 melanoma cell lines derived from metastatic melanomas alsoexpressed high levels of ICAM-1 and DAF (data not shown). The finding ofhigh level ICAM-1 expression on all the metastatic melanoma cells testedsupports several reports in the literature noting increased levels ofICAM-1 expression in vivo correlates with increased metastatic ability(Johnson J P, et al: 1988; Kageshita T, et al: 1993; Miller B E andWelch D R: 1990; Natalie P G, et al: 1997).

EXAMPLE 2

2.1. Infection of Melanoma Cell Lines by CAV21

Monolayers of two culture-adapted melanoma cell lines Miller and MM200were infected with CAV21 prepared in Example 1 at a multiplicity ofinfection of 1.0 for 1 hour prior to removal of the inoculum and thecells incubated in culture medium (DMEM containing 1% foetal calf serumand penicillin streptomycin) for 24 hours at 37° C. The results shown inFIG. 3 indicate that CAV21 was able to induce significant changes in thecellular cytopathology of both cell lines as early as five hours postinfection (PI) and by nine hours PI almost complete killing of all themelanoma cells.

EXAMPLE 3

3.1. Infection of Melanoma Cells from Primary Melanoma by CAV21

Cells from a primary melanoma removed from a nude mouse that had beenpreviously subcutaneously inoculated with human melanoma cells from cellline ME 4405 using conventional methods, were highly susceptible toCAV21 infection and killing, even at a challenge rate of 0.005 CAV21particles per melanoma cell as shown in FIG. 4.

EXAMPLE 4

4.1. Infection of Melanoma Cells Isolated From Tissue Biopsy by CAV21

Melanoma cells were isolated from fresh biopsy of a primary chest wallmelanoma by the “spilling” technique and by digestion incollagen-trypsin and DNAase. Briefly, cells were released from themelanoma biopsy by macerating the biopsy with the plunger of a 10 mlsyringe. The resulting melanoma cell suspension was purified on aFicol-Hypaque (Amersham Pharmacia, Uppsala, Sweden) gradient.Contaminating fibroblasts and leucocytes were removed by mixing withDynal beads coated with monoclonal antibodies (Mab's) to humanfibroblasts (Cat#; MASS 16X, SeraLab) and to the leucocyte commonantigen (CD45, Cat#17-0804-3, Amrad Biotech, Victoria, Australia).

Subsequently, 1×10⁶ cells were placed into wells of a 24-well tissueculture plate and inoculated with approximately 1×10⁵ plaque formingunits of CAV21 prepared in Example 1. Following incubation at 37° C. for20 hours, cells were assessed for cell death by staining with propridiumiodine and microscopic analysis.

FIG. 5 shows that both adherent and suspension primary melanoma cellswere efficiently killed as a result of CAV21 infection during the 20hour incubation period.

EXAMPLE 5

5.1 Expression of ICAM-1 and DAF on Melanoma Cells Susceptible to CAV21Infection

To confirm melanoma cells are highly susceptible to infection andresultant killing by CAV21, six additional human melanoma cell linesderived from primary human melanomas were infected with CAV21 preparedin Example 1.

FIG. 6(A) indicates that all melanoma cell lines except one (ME 105)were killed as a result of CAV21 infection during a 23 hour incubationperiod.

To confirm high level expression of ICAM-1 and DAP on the surface ofmalignant melanoma cells, cells from each cell line were treated withthe Mab IH4 and Mab WEHI-CAM. The binding of the anti-DAF andanti-ICAM-1 Mab was detected by flow cytometric analysis as describedabove. The fluorescence histograms shown in FIG. 6(B) confirm high levelexpression of DAF and ICAM-1 on the surface of all melanoma cell linesexamined except the ME 105 cell line. The lack of DAF and ICAM-1expression rendered this cell line retractile to CAV21 infection.

EXAMPLE 6

6.1 Selective Infection of Melanoma Cells Expressing ICAM-1

To highlight the selective nature of CAV21 infection of ICAM-1expressing human melanoma cells, monolayers of melanoma cell line MM 200were inoculated with approximately 1×10⁵ plaque forming units of CAV21,Coxsackievirus B3 (CVB3), Echovirus type 7 (E7) or Coxsackievirus B1(CVB1) in wells of a 24-well tissue culture plate for one hour at 37°C., respectively. The viral inoculate was subsequently removed and thecell monolayers then washed with phosphate buffered saline (PBS), and1.0 ml of DMEM containing 1.0% foetal calf serum was added to each welland the cells incubated at 37° C. for 48 hours. To quantitate cellsurvival, monolayers were incubated with a crystal violet/methanolsolution, washed with distilled water and microscopically examined at100×.

FIG. 7 shows that following the 48 hour incubation period only CAV21infected the MM 200 melanoma cells while the reverse occurred in therhabdomyosarcoma cells (RD) where CVB 1, CVB3 and E7 infection andkilling is evident. RD cells express DAF but no ICAM-1. However, whenICAM-1 is expressed on the surface of RD cells they are highlysusceptible to CAV21 induced infection and killing.

EXAMPLE 7

7.1 Infection of Melanoma Biopsy With CAV21

Sections of solid human melanoma lymph node biopsies were placed inwells of a 24-well tissue culture plate and mock infected or challengedwith approximately 1×10⁵ plaque forming units of CAV21 or CVB3.

The results shown in FIG. 8 indicate that CAV21 infection resulted insevere tissue destruction around the perimeter of the melanoma biopsytreated with that virus while no detectable viral membrane destructionwas observed in the mock and CVB3 infected biopsies.

EXAMPLE 8

8.1. Lytic Infection of Human Melanoma Cells by CAV21 and CAV15

To assay the oncolytic potential of CAV15 and CAV21 on human melanomacell lines, Sk-Mel-28 and ME4405 cells were seeded into flat-bottom96-well microtiter plates (Becton Dickinson) at 3×10⁴ cells per well.Following incubation for 24 h at 37° C., culture medium was removed andreplaced with fresh medium containing the appropriate viral serialdilution in a final volume of 100 μl. Stock viral preparations wereserially diluted 10⁻¹ through to 10⁻⁷. Following viral inoculation, theplates were incubated at 37° C. for 48 h and cell survival was detectedby crystal violet staining as described above.

All three cell lines RD-ICAM-1, Sk-Mel-28 and ME4405 were found to bepermissive to lytic infection by both CAV21 and CAV15. Following anincubation period of 48 h, the no virus control showed no signs of viralinduced CPE while extensive cell lysis was observed across all cellcultures at a dilution of 10⁻¹ and 10⁻². At higher viral dilutionsSk-Mel-28 cells were shown to be more permissive to viral lysis comparedto ME4405 and RD-ICAM-1 cell lines.

The overall oncolytic potential of CAV21 and CAV 15 was higher in themelanoma cell lines, compared to the control RD-ICAM-1 cells. While allcell types express similar levels of ICAM-1, DAF expression in RD-ICAM-1cells is significantly lower than on melanoma cells (see FIG. 2)accounting for lower viral attachment via DAF to RD-ICAM-1 cells. DAFhas previously been shown to be a low affinity sequestration moleculefor many Coxsackieviruses, assisting the capture of virus particles andhence infectivity of the cells (Lea S M, et al; 1998). The presence ofhigher levels of DAF expression on the melanoma cell lines compared tothe RD-ICAM-1 cells increases the probability of viral access to ICAM-1receptors, thus leading to an increased level of infection and celllysis.

8.2. Lytic Infection of Human Prostate Cancer Cells by Coxsackievirus

Cells from the human prostate cancer cell line CP3 (which expressesICAM-1) were seeded into a flat-bottom 96-well microtitre plate (BectonDickenson) at 3×10⁴ cells per well and treated with serial dilutions ofCAV13, CAV15, CAV21 and the Coxsackievirus B-group virus CVB3 followingincubation of the cells, as described in Example 8.1 above. PC3 cellsare available from the American Type Culture Collection (ATCC) Manassas,Va., USA under Accession No. CRL-1435.

As shown in FIG. 9, the PC3 cells were highly permissive to lyticinfection by CAV15. Extensive lytic infection was also observed for bothCAV13 and CAV21.

8.3. Selective Replication of CAV21 and CAV15 in the Human Melanoma CellLines Sk-Mel-28 and ME4405

The selectivity of CAV21 and CAV15 for the melanoma cell lines Sk-Mel-28and ME4405 was studied using an in vitro specificity assay.

Sterile cell culture inserts were used to divide the wells of a standardsix well plate tissue culture plate. Inside the cell culture insert,either Sk-Mel-28 cells or ME4405 cells were grown, with MRC5 or RD cellsgrown around the cell culture insert. Once the cells had adhered, thecell culture inserts were removed from each of the well allowing thecell culture media to evenly cover the co-culture. When the perimetersof both cell populations had fused, the co-cultures were washed twicewith PBS and then inoculated with 500 μl of either PBS or stock virus(10⁵ TCID₅₀) for 1 h at 37° C. Following incubation at 37° C., freshDMEM containing 1% FCS was added to each of the wells and the platesincubated for 48 h at 37° C. in a 5% CO₂ atmosphere. Cell monolayerswere monitored by light microscopy for signs of virus-induced CPE, priorto each well being stained with 3 ml of crystal violet solution for thedetection of cell survival from viral induced lytic infection. Thecapacity of CAV21 and CAV15 viruses to specifically lytically destroymelanoma cells without infecting non-melanoma surrounding cells isillustrated in FIG. 10.

As can be seen, the inner cultures of melanoma cells in each welltreated with CAV21 or CAV15 were totally destroyed by the viruses, butwere unaffected by CVB3 virus which does not employ ICAM-1 as a receptorfor cell entry. CVB3 which employs the Coxsackie- and adenovirusreceptor (CAR) for cell entry (10). MRC5 cells appeared to be refractoryto lytic infection by both CAV21 and CAV15. These cells are derived froma human lung fibroblast culture and only express low levels of ICAM-1(unpublished data). The present data shows that rapid and effectivelytic infection of target cells facilitated high level ICAM-1 and DAFexpression. RD cells, which do not express ICAM-1, were not destroyed byeither CAV21 or CAV15 infection. Furthermore, the results show little ifany spread of CAV21 and CAV 15 to receptor negative cells that are indirect contact with virally infected receptor-bearing cells.

EXAMPLE 9

The lytic infection of preformed melanoma tumours in vivo was evaluatedby a series of animal challenge experiments using NOD-SCID mice.

9.1. Development of Melanoma Xenografts in NOD-SCID Mice

All animal work was performed under guidelines approved by TheUniversity Of Newcastle Animal Care and Ethics Committee. NOD-SCID micewere housed in pathogen-free quarters in the animal handling facilitylocated at the David Maddison Building, Level 5, Newcastle, NSW,Australia.

Sk-Mel-28 and ME4405 cells were grown in DMEM containing 10% FCS. Thecells were harvested and washed twice with DMEM, and resuspended insterile PBS. The cell concentration of the suspension was determinedwith a haemocytometer and cell viability was assessed by trypan bluestaining. Only cell preparations with >95% viability were used forxenotransplantation. Prior to xenotransplantation, animals wereanaesthetised with intraperitoneal (i.p) injections of Rompun/Ketamine(50 mg/kg). For the monitoring of animals and measurement of tumourgrowth, animals were anaesthetised with 3% isofluorane.

The tumour cells were xenografted into the flank of anaesthetised 4-6week old female NOD-SCID mice. Xenograft tumour growth was observeddaily and measured with callipers at various intervals with allmeasurements recorded in millimetres over the course of 5 weeks.Estimates of tumour volumes were calculated using known methods (DaviesC D, et al; 1997).

9.2. Subcutaneous Viral Delivery

In a preliminary experiment employing fifteen NOD-SCID mice, the localsubcutaneous delivery of virus through ex vivo infected cells wasassessed for inhibition of tumour growth. The mice in the control group(n=5) were injected subcutaneously with Sk-Mel-28 cells (1×10⁷) cells atindividual sites in both the upper and lower flank. The CAV21 group(n=5) received an injection of 1×10⁷ Sk-Mel-28 cells in the upper flankand a second injection of Sk-Mel-28 (1×10⁷) cells that had beenpre-incubated with 10⁴ TCID₅₀ of CAV21 at room temperature for 1 hour exvivo. The CAV15 group (n=5), was treated the same as the CAV21 groupexcept that the second injection in the lower flank contained Sk-Mel-28(1×10⁷) cells that had been incubated with 10⁴ TCID₅₀ of CAV15. Fourweeks post-injection, a representative of the control group wassacrificed and shown to bear two individual tumour masses correspondingto the two injections sites of the Sk-Mel-28 (1×10⁷) cells. In contrasta representative of the CAV21 group beared no detectable tumourformation in either the uninfected cell or virally infected cell sitesof injection (FIG. 11). Upon autopsy examination, all remaining membersof the control group were shown to possess two distinct melanomaxenograft tumour growths, while remaining members of the CAV21 group (17weeks post injection) exhibited no detectable tumour growth in eithersite of injection. Mice in the CAV15 group exhibited no tumour formationat 4 weeks post-injection.

9.3. Intratumoural Viral Delivery

Twenty NOD-SCID mice were injected with Sk-Mel 28 cells (1×10⁷) in theupper flank. When the tumour volume reached ˜50-100 mm³ the animals wererandomly divided into groups of five and housed in separate cages.Groups of mice were injected intratumourally with 100 μl of active CAV21or CAV15 containing 10^(3.2) or 10^(4.2) TCID₅₀ doses, respectively. Theremaining animals received 100 μl of PBS injected directly into thexenografts. The different treatment groups were housed in individuallyvented cages maintained under negative pressure, ensuring that virus andother pathogens were contained within the individual cages.

A dose of 10^(3.2) or 10^(−4.2) TCID₅₀ of either CAV21 or CAV15respectively, was sufficient to produce significant tumour reduction inanimals bearing preformed Sk-Mel-28 tumours at 14 days post-injection.The trend of reduction of tumour burden continued for the next 14-21days. No detectable tumours were observed at 30-35 days post-injection(see FIGS. 12 and 13). The difference observed between the CAV21 treatedgroup and the PBS treated control group was statistically significant(P=0.0023, t test). Animals bearing Sk-Mel-28 tumours and injected withCAV21 showed no clinical signs of CAV21 illness. The capacity of CAV15to drastically reduce melanoma tumour burden is shown in FIG. 14. At 35days post-injection, the melanoma xenograft treated with PBS wasapproximately 2037 mm³ while the CAV15 treated tumour was approximately2 mm³ in volume (P=0053, t test). The CAV15 treated tumour showncomprises mostly residual connective tissue.

9.4. Intratumoral Delivery of CAV21 to ME4405 Xenograft

The intratumoural delivery of CAV21 to a different melanoma (ME4405)xenograft was undertaken to further confirm the anti-tumour therapypotential of this virus. Fifteen NOD-SCID mice were injected with ME4405cells (5×10⁶) subcutaneously in a single site on the flank. When tumourvolumes had reached approximately 500 mm³, the animals were randomlydivided into groups of five and housed in separate cages. Five animalswere injected intratumourally with 100 μl of active CAV21 containing1032 TCID50 doses, while five mice received 100 μl of PBS injecteddirectly into the xenografts and the remaining five mice were leftuntreated. As shown in FIG. 15, intratumoural administration of CAV21was able to markedly reduce tumour development of ME4405 cells within 25days post-injection even though the initial pre-injection tumour volumewas 5-fold greater than those utilised above. The ME4405 xenografts wereobserved to be more aggressive than the Sk-Mel-28 tumours as assessed bysignificantly faster growth rates of tumours in the control groups.

The ME4405 cell line generated highly vascular aggressive tumourscompared to Sk-Mel-28 tumours which grew at a slower rate and were notas vascular as the ME4405 tumours.

In contrast to mice bearing Sk-Mel-28 xenografts, when CAV21 wasinjected into animals with ME4405 tumours, some signs of illness wereobserved, the most notable being a transient weakness in both the foreand hind limbs. No positional abnormalities were observed.

9.5. Discussion of Results

This study demonstrates that CAV13, CAV15 and CAV21 have the capacity tolytically destroy malignant cell lines.

Specifically, the in vitro analysis of CAV21 and CAV15 infection ofmelanoma cells shows that these two viruses are able to selectivelyinfect Sk-Mel-28 and ME4405 cell lines as a result of the expression ofICAM-1 and DAF while each of the Coxsackieviruses mentioned above wereable to infect and cause the death of cells of the prostate cancer linePC3. Moreover, the intratumoural injection of CAV21 and CAV15 intoxenografts of human melanoma cell lines grown in the flanks of NOD-SCIDmice show that CAV21 and CAV15 possess therapeutic applications againstmalignant melanoma. The direct injection of either of the two virusesinto pre-formed melanoma tumours suppressed tumour growth and led tosignificant tumour regression and in some cases complete tumourdestruction compared to control animals. Furthermore, the delivery ofcells infected by virus ex vivo yielded total inhibition of tumourgrowth and demonstrates that ex vivo CAV21 infected melanoma cells arecapable of delivering sufficient virus to inhibit local tumour growth.In addition, injection of infected cells subcutaneously in a distantregion to the initial tumour challenge shows that the virus can travelsystemically.

The pathogenesis of CAV21 and CAV15 infections are mainly asymptomaticor manifest by no more than minor malaise. The Coe strain of CAV21 hasrecently been approved for live administration by the Food and DrugAdministration (FDA) of the United States of America for the clinicalassessment of specific anti-viral agents against CAV21 (90). The recentdevelopment of specific antiviral agents against CAV21 and CAV 15provides the added safety precaution of drug intervention to controlviral infection.

Although the present invention has been described hereinbefore withreference to a number of preferred embodiments, the skilled addresseewill understand that numerous modifications and variations are possiblewithout departing from the scope of the invention.

References Cited:

1. Kageshita T, Yoshii A, Kimura T, Kuriya N, Ono T, Tsujisaki M, Imai Kand Ferrone S (1993). Clinical relevance of ICAM-1 expression in primarylesions and serum of patients with malignant melanoma. Cancer Res.October 15; 53(20):4927-32.

2. Kraus A, Masat L and Johnson J P (1997). Analysis of the expressionof intercellular adhesion molecule-J and MUC18 on benign and malignantmelanocytic lesions using monoclonal antibodies directed againstdistinct epitopes and recognising denatured, non-glycosylated antigen.Melanoma Res. August 7; Suppl 2:S75-81.

3. Morandini R, Boeynaems J M, Hedley S J, MacNeil S and Ghanem G(1998). Modulation of ICAM-1 expression by alpha-MSH in human melanomacells and melanoxytes. J Cell Physiol. June; 175(3):276-82.

4. Staunton D E, Merluzzi V J, Rothlein R, Barton R, Marlin S D andSpringer T A (1989). A cell adhesion molecule, ICAM-1 is the majorsurface receptor for rhinoviruses. Cell. 56:849-853.

5. Cheung N K, Walter E I, Smith-Mensah W H, Ratnoff W D, Tykocinski M Land Medof M E (1998). Decay-accelerating factor protects human tumorcells from complement-mediated cytotoxicity in vitro. J Clin Invest.April; 81(4): 1122-8.

6. Nemunaitis J (1999). Oncolytic viruses. Investigational New Drugs17:375-386

7. Fenner F, McAuslan B R, Mims C A, Sambrook J and White D O. TheBiology of Animal Viruses. Academic Press, New York, 1974 Second Ed.

8. Alemany R, Gomez-Manzano C, Balague C, Yung W K, Curiel D T, KyritsisA P and Fueyo J (1999). Gene therapy for gliomas: molecular targets,adenoviral vectors, and oncolytic adenoviruses. Exp Cell Res. 252:1-12.

9. Andreansky S S, He B, Gillespie G Y, Soroceanu L, Markert J, Chou J,Roizman B and Whitley R J (1996). The application of geneticallyengineered herpes simplex viruses to the treatment of experimental braintumors. Proc Natl Acad Sci USA 93:11313-8.

10. Coffey M C, Strong J E, Forsyth P A and Lee P W K (1998). Reovirustherapy of tumours with activated Ras pathway. Science. 282:1332-1334.

11. Strong J E, Coffey M C, Tang D, Sabinin P and Lee P W K (1998). Themolecular basis of viral oncolysis: usurpation of the Ras signallingpathway by reovirus. 17(12):3351-3362

12. Randazzo B P, Kesari S, Gesser R M, Alsop D, Ford J C, Brown S M,Maclean A and Fraser N W (1995). Treatment of experimental intracranialmurine melanoma with a neuroattenuated herpes simplex virus 1 mutant.Virology 211:94-101.

13. Satyamoorthy K, Soballe P W, Scans F and Herlyn M (1997). Adenovirusinfection enhances killing of melanoma cells by a mitotoxin. CancerResearch 57:1873-1876.

14. Hemmi S, Geertsen R, Mezzacasa A, Peter I and Dummer R (1998). Thepresence of human Coxsackievirus and adenovirus receptor is associatedwith efficient adenovirus-mediated transgene expression in humanmelanoma cell cultures. Human Gene Therapy 9:2363-2373.

15. Shafren D R, Dorahy D J, Ingham R A, Burns G F and Barry R D (1997).Coxsackievirus A21 binds to decay-accelerating factor but requiresintercellular adhesion molecule 1 for cell entry. J. Virol. June;71(6):4736-43.

16. Flint S J, Inquest L A W, Krug R M, Racaniello V R and Skalka A M(2000). Principles of virology: molecular biology, pathogenesis, andcontrol. ASM Press, Washington.

17. Marshall J F and Hart I R (1996). The role of αv-integrins in tumourprogression and metastasis.

18. Bjørge L, Jensen T S and Matre R (1996). Characterisation of thecomplement-regulatory proteins decay-accelerating factor (DAF, CD55) andmembrane cofactor protein (MCP, CD46) on a human colonic adenocarcinomacell line. Cancer Immunol Immunother. 42:185-192.

19. Nasu R, Mizuno M, Kiso T, Shimo K, Uesu T, Nasu J, Tomoda J, Okada Hand Tsuji T (1997). Immunohistochemical analysis of intercellularadhesion molecule-1 expression in human gastric adenoma andadenocarcinoma Virchows Arch 430:279-283.

20. Koyama S, Ebihara T and Fukao K (1992). Expression of intercellularadhesion molecule 1 (ICAM-1) during the development of invasion and/ormetastasis of gastric carcinoma. J. Cancer Res. Clin. Oncol.118:609-614.

21. Rokhlin O W and Cohen M B (1995). Expression of cellular adhesionmolecules on human prostate tumor cell lines. Prostate. April;26(4):205-12.

22. Sgagias M K, Nieroda C, Yannelli J R, Cowan K H and Danforth Jr. D N(1996). Upregulation of DF3, in association with ICAM-1 and MHC class IIby IFN-gamma in short-term human mammary carcinoma cell cultures. CancerBiother Radiopharm. 11:177-85.

23. Regidor P A, Callies R, Regidor M and Schindler A E (1998).Expression of the cell adhesion molecules ICAM-I and VCAM-I in thecytosol of breast cancer tissue, benign breast tissue and correspondingsera. Eur J Gynaecol Oncol. 19:377-83.

24. Bacuss S S, Zelnick C R, Chin D M, Yarden Y, Kaminsky D B,Bennington J, Wen Ds Marcus J N and Page D L (1994). Medullary carcinomais associated with expression of intercellular adhesion molecule-1.Implication to its morphology and its clinical behaviour. Am J Pathol.December; 145(6):1337-1148.

25. Maloney D G, Donovan K and Hamblin T J (1999). Antibody therapy fortreatment of multiple myeloma. Seminars in Hematology. 36 (1 Suppl3):30-33.

26. Fernandez-Real J M, Villabona C, Femandez-Castaner M, Sagarra E,Gomez-Saez J M and Soler J (1996). Expression of ICAM-I in distantmetastatic thyroid carcinoma. J Endocrinol Invest. March; 19(3):183-185.

27. Natalie P G, Hamby C V, Felding-Habermann B, Liang B, Nicotra M R,Di Filippo F, Giannarelli D, Temponi M, Ferrone S (1997). Clinicalsignificance of alpha(ν) beta3 integrin and intercellular adhesionmolecule-1 expression in cutaneous malignant melanoma lesions. CancerRes. April 15; 57(8):1554-60.

28. Reed L J and Muench H A (1938). A simple method of estimating fiftypercent endpoints. Am J Hyg. 27:493-497.

29. Berendt A R, McDowall A, Craig A G, Bates P A, Sternberg M J E,Marsh K, Newbold C I and Hogg M (1992). The binding site on ICAM-1 forPlasmodium falciparum-infected erythrocytes overlaps, but is distinctfrom the LFA-1 binding site. Cell. 68:71-81.

30. Johnson J P, Stade B G, Hupke U, Holzmann B, Schwable W andReithmuller G (1988). The melanoma progression-associated antigen P3.58is identical to the intercellular adhesion molecule ICAM-1. Immunology.178:275-284.

31. Miller B E and Welch D R (1990). Intercellular adhesion molecule-1(ICAM-1) expression by human melanoma cells; association with leukocyteaggregation and metastatic potential. Clin. Exp. Metastasis. 8:80.

32. Lea S M, Powell R M, McKee T, Evans D J, Brown D, Stuart D I and vandeer Merowe P A (1998). Determination of the affinity and kineticconstants for the interaction between the human virus echovirus II andits cellular receptor, CD55. J. Biol. Chem. 273:30443-60447.

33. Davies C A L, Muller H, Hagen I, Gareth M and Helton M H (1997).Comparison of extracellular matrix in human osteosarcomas and melanomasgrowing as xenografts, multicellular spheroids and monolayer cultures.Anticancer Research. 17:4317-4326.

What is claimed is:
 1. A method of treating abnormal cells in a mammal,comprising administering to the mammal an effective amount of a humanEnterovirus-C (HEV-C) that binds to intercellular adhesion molecule-1(ICAM-1) for infectivity of the abnormal cells and is thereby capable ofinfecting the abnormal cells whereby death of the cells is caused,wherein the abnormal cells are cancer cells, and further wherein theHEV-C is administered intravenously, intratumorally, intraperitoneallyor intramuscularly.
 2. The method of claim 1, wherein the HEV-C binds toboth ICAM-1 and decay accelerating factor (DAF) for infectivity of theabnormal cells.
 3. The method of claim 1, wherein expression of ICAM-1is upregulated on the abnormal cells relative to normal said cellsexpressing their normal phenotype.
 4. The method of claim 1, whereinexpression of ICAM-1 is upregulated on the abnormal cells relative tocells of surrounding tissue in which the abnormal cells are found. 5.The method of claim 1, wherein the HEV-C is a Coxsackievirus.
 6. Themethod of claim 1, wherein the HEV-C is a recombinant virus.
 7. Themethod of claim 1, wherein the abnormal cells are cells of a malignancyselected from the group consisting of a malignancy of the skin,melanoma, multiple myeloma, head and neck cancer, prostate cancer,stomach cancer, breast cancer, gastric cancer, colorectal cancer,glioma, and colon cancer.
 8. The method of claim 1, wherein the HEV-C isadministered by injection.
 9. The method of claim 1, wherein the HEV-Cis administered to the mammal in a dosage greater than about 1×10²plaque forming units per ml of inoculant.
 10. The method of claim 9,wherein the HEV-C is administered to the mammal in a dosage of betweenabout 1×10² to 1×10¹ plaque forming units per ml of the inoculant.